Given our interest in the APC/C and its downstream targets, we have been focusing our studies on an indirect target of the APC/C. Separase is the protease that cleaves the cohesin complex that holds homologs together at meiosis. Securin inhibits separase from carrying out this role until the metaphase to anaphase transition, at which time securin is ubiquitinated by the APC/C and degraded by the 26S proteasome. We have taken a genetic approach to identify regulators and substrates of separase. We have three mutant alleles of sep-1 and have recently shown, through a collaboration with Dr. Joshua Bembenek, that these mutants all have defects in cortical granule exocytosis (CGE). CGE is important for the secretion of components necessary for eggshell formation, which is indirectly required for proper polar body extrusion. In order to identify other genes that function in the separase pathway, we carried out a suppression screen with a temperature-sensitive allele of sep-1, e2406ts. We have identified three suppressors that restore viability to sep-1 mutants at the non-permissive temperature. One of these mutants is an intragenic suppressor;the other two are extragenic. We have recently determined that one of these suppressor mutations is in a phosphatase gene called pph-5. This phosphatase mutant, in an otherwise wild-type background, has no obvious phenotypes on its own. Our pph-5 allele, av101 (and a two deletion alleles of the pph-5 gene), suppresses the embryonic lethality of two of our three sep-1 alleles. RNAi of pph-5 also suppresses the embryonic lethality of two of our three sep-1 alleles. RNAi depletion or genetic mutation of pph-5 restores CGE to wildtype levels in the suppressed sep-1 backgrounds. In collaboration with Harold Smith (NIDDK), we have also carried out deep sequencing to determine the mutation in our second extragenic suppressor, av102. A mutation was found in a gene that encodes a carboxyl methylase. Orthologs of this gene in yeast are known to promote the formation of the protein phosphatase 2A complex. Thus our studies in C. elegans suggest that two distinct phosphatases may regulate separase function. Since numerous components of the separase pathway are known kinases or phosphoproteins, the removal of specific phosphate groups must play an important regulatory role in separase activity. We seek to determine the substrates of these two phosphatases and the mechanisms by which mutations suppress sep-1 lethality.